Carnitine palmitoyltransferase I activity monitoring in fibroblasts and leukocytes using electrospray ionization mass spectrometry.
Carnitine palmitoyltransferase I (CPT I) is one of the enzymes associated with normal mitochondrial membrane transport of certain metabolites. The importance of the enzyme in normal energy production is well illustrated during fasting conditions when a large flux of long-chain fatty acids must be transported over the mitochondrial membrane to undergo beta-oxidation. Up to now CPT I activity has been assayed in various tissues, including liver, leukocytes, platelets, and fibroblasts by the use of an isotope exchange forward assay which measures the rate of palmitoyl-l-[methyl-3H]carnitine formation from palmitoyl-CoA and l-[methyl-3H]carnitine. We have developed an electrospray ionization mass spectrometric method for detecting palmitoylcarnitine formation from palmitoyl-CoA and carnitine, thus avoiding the use of radiolabeled isotopes. In this assay, time-dependent conversion of free carnitine by CPT I to palmitoylcarnitine is measured quantitatively, relative to isotopically labelled palmitoylcarnitine, by parent ion monitoring of fragment ion m/z 85. The specific activity of CPT I in fibroblasts and leukocytes compared well with the activity determined with the isotope exchange method, however, the combination of high sensitivity and selectivity of tandem mass spectrometry along with the environment-friendly nature of the electrospray method makes it an ideal technique to measure CPT I activity.
Nolte
L
L
Department of Biochemistry and Microbiology, Potchefstroom University for Christian Higher Education, South Africa.
van der Westhuizen
F H
FH
Pretorius
P J
PJ
Erasmus
E
E
D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D013058:Mass Spectrometry / Q000379:methods; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010172:Palmitoylcarnitine / Q000378:metabolism
D010171:Palmitoyl Coenzyme A; D010172:Palmitoylcarnitine; D002334:Carnitine O-Palmitoyltransferase